Latest revision as of 02:19, 18 October 2018

Design

Engineering fungal mycelium to create viable building materials for Mars is not a small challenge. This project, which was inspired by conversations with Lynn J. Rothschild from NASA, and the Stanford-Brown-RISD iGEM team, whom we owe thanks to both for the inspiration and subsequent sparring on ideas, has relied on several scientific fields to try to tackle the problems we have found along the way. Being inspired by the idea of simple and cost-effective construction on Mars, our design focuses on the fungal aspect of how a cyanobacteria/fungal production system can be used to create building materials. As we explored this idea, we were presented with many problems to be solved and even more, questions to be answered.
The first and most pressing one, how does one cultivate a fungus on Mars? Moreover, which fungal species should we use? Can we determine and model the growth characteristics of our fungi? Is the strength of fungal materials determined by any distinct genes and is it possible to regulate these in a way that will make our materials even tougher? And not least, how do we make the system cost-effective? We set out to answer these questions to guide the creation of our final design.

Growing Fungus on Mars

Growing live fungus on Mars will be challenging as the environment on Mars is extremely hostile compared to Earth. In addition to the extreme cold, the atmosphere is very thin at less than 1% of the density of Earth’s atmosphere and consists of 96% carbon dioxide and virtually no oxygen (1). Moreover, our fungus needs organic compounds as a source of carbon and energy. Bringing substrates from Earth is out of the question due to the costs of transportation, thus in situ production of materials is vital. To solve the problem of available carbon sources, one can introduce cyanobacteria to the production system. Cyanobacteria can utilize the carbon dioxide in the Martian atmosphere, albeit, like the fungus, they, too, are unable to grow directly in the extreme conditions of Mars. To solve the problem of the Martian hostility it is necessary to have a production facility. Such a production facility for cyanobacteria has been theorized by researchers at NASA (2). Using covered thermally insulated ponds with compressed air and water from the Martian ice, it would be possible to grow cyanobacteria on Mars. Supporting this idea is the fact that all of the essential elements needed for cyanobacterial and even plant growth can be found on Mars (2). Based on this system theorized by NASA, we suggest appending it with the capabilities of using the cyanobacterial biomass to produce fungal materials.

Fig 1: - Creating bricks in unconventional molds.

However, non-destructive ways of harvesting nutrients could possibly lead to a more effective process. So instead of using lysed-dead cyanobacteria, there is also the possibility of using biomass produced by them as a secreted substrate. Cyanobacteria can provide heterotrophic organisms with both nitrogen and oxygen through their photosynthetic and nitrogen-fixing pathways. Specifically, they can convert carbon dioxide, the most abundant gas in Mars’ atmosphere, into oxygen and at the same time provide NH₄⁺ to the feeding organisms by utilizing N fixation (2). These problems are beyond the scope of our project, but provides an interesting starting point for future projects.

Choice of Organism

The choice of organism used in our project was based on an inductive strategy. We had two variables needed to be fulfilled in our attempt to achieve the goals we had set. The first was to find a fungus species that, given the right substrate, would have the ability to form firm brick structures. The second was to use a safe microorganism with a sequenced genome and established genetic methods, thus enabling us to do genetic modifications. The project started with three candidate-organisms: Pleurotus ostreatus, Schizophyllum commune and Aspergillus oryzae. These organisms have previously been grown on a variety of substrates and some have been used for creating bricks. We were also recommended to use a Fusarium by researchers at Novozymes, but opted to focus on the ones we had multiple recommendations on. For various reasons, our choice ended up being A. oryzae; A. oryzae is comparatively easy to grow on various substrates, it grows fast, gives consistently good results and is a well-established model organism. Though the other fungal species have previously shown to have desirable characteristics, when it comes to creating fungal materials, the results of our initial experiments made us decide on doing our research in A. oryzae with the possibility of transferring the discoveries to other fungal species at a later point in the project.

How to Build Structures on Mars

When wanting to erect livable habitats on Mars made of fungal materials, an obvious question is: What physical parameters will the fungal bricks have to withstand for them to be used as building material on Mars? Martian gravity is about 38% of Earth’s (3) makes it easier to erect self-sustainable structures on Mars than on Earth. However, the greater challenge is enabling structures to sustain the internal pressure generated by the pressure difference between the liveable habitat and that of the Martian atmosphere. The average pressure on Earth is 1.013 bar with 21% oxygen. The lowest human life-sustaining pressure is 0.121 bar, assuming a 100% oxygen concentration (4). Though, this is not desirable due to extreme fire hazards. At these oxygen concentrations, everything becomes combustible. Near the lower border of what is safe/healthy and sustainable for human life is an atmosphere containing 19.5% oxygen at approximately 0.65 bar (5). As stated earlier, the average Martian pressure is 0.006 bar. This is only 0.6% of the average barometric pressure on earth (6).

What building design/geometry is the best at containing pressures? For simulation purposes, the internal pressure is 1 atmosphere. Humans can live at lower pressures, but lab-equipment and plant growth are sensitive to lower pressures (7). This is why the International space station maintains an internal pressure of 1 ATM. Looking at a dome and a box design, the COMSOL simulations on Fig(2) and Fig(3), strongly indicate, that the dome design is the better choice. We assume that: Equal internal volume, an internal pressure of 1 bar and an external pressure of 0.006 bar. In order for the box, to be able to contain this pressure, the wall thickness would have to be 1.3 m thick, compared to the domes 0.3 m wall thickness. The pressure distribution is also clearly more uniformly distributed in the dome, compared to the box. The relations between “volume to area” of the dome and box are as follows:
\begin{equation} Area_{circle} = r^2 \cdot \pi \end{equation} \begin{equation} Area_{dome} = Area_{circle} + \frac{r^2 4 \pi}{2} = 3 r^2 pi \end{equation} \begin{equation} Area_{box} = 2a^2 + 2a^2 + 2a^2 =6 a^2 \end{equation} \begin{equation} \frac{Area}{Volume} -relations : \end{equation} \begin{equation} \frac{a^3}{Area_{box}} = a \cdot \frac{1}{6} \end{equation} \begin{equation} \frac{\frac{4}{3} \pi r^ 3}{Area_{dome}} = \frac{2}{9} \cdot r \end{equation} What becomes apparent from this, is that the “volume to area”- relation is lower for a box. This means that you would need more building material to construct a box, compared to a dome of equal volume.

Fig 2: - The picture shows how the internal stresses accumulate in a dome, simulated in COMSOL. It also shows the minimal wall thickness (0.3 m) when using Aspergillus as building material in Mars, if the dome has to be able to contain 1 bar.

Fig 3: - The internal stress accumulation of a box is a lot higher than a dome. In order for an box, of same volume as Fig(2), the wall thickness would have to be 1.3 m in order to contain 1 bar against the Martian atmosphere.

Improving the fungal materials through synthetic biology

When using fungal mycelium and organic materials as the basis for building materials, a new venue of possibilities suddenly appear. Fungi are genetically modifiable. This allows for tuning of different external parameters or even adding properties such as biosensors etc. Thus, the interesting question to answer in this aspect is; what genetic modifications are beneficial when it comes to construction on Mars?

Like any other organism, any fungus is optimized for the survival of its species in its niche. No fungal strain is solely optimized for creating strong materials, as that would not be an advantageous evolutionary strategy. By making the fungus a little worse at surviving, we aim to make it better for a building. In our project, we ended up focusing primarily on two genes; melA and gfaA.

Protection from UV radiation and Melanin

One part of what makes the surface of Mars a hostile environment is its radiation. Unlike the terrestrial atmosphere, the Martian atmosphere does not shield well against cosmic radiation, making radiation a serious concern on the red planet (10). Part of the radiation issue is short wavelength UV radiation such as UVB and UVC, which is not just harmful to living tissue but also causes radiation damage to solid materials (11). Nature has evolved systems that give the protection against UV radiation, most well-known is the pigment melanin. Overexpressing melanin might make fungal materials more resilient against radiation damage. Studies have shown that melanotic bacteria experience increased protection from the full spectrum of UV compared to counterparts without melanin (12). To slow down the potential erosion from UV radiation, we wanted to express the gene melA from Rhizobium etli CFN42 (13) in our fungal host. A previous iGEM team from the Technical Institute of Tokyo have shown that overexpression of melanin in E. coli can been achieved using the melA gene. melA encodes this for a tyrosinase that catalyses the enzymatic steps converting tyrosine into dopaquinone. We investigated the biosynthetic pathway of melanin in A. oryzae and discovered the same metabolic step as encoded for by melA (14). Heterologous expression of MelA in A. oryzae may thus also increase the production of melanin.

Melanin (Codon optimized for A. oryzae)

As a part of the project we set out to codon optimize, the Tokyo Tech melA gene from Rhizobium etli CFN42 to increase the expression in A. oryzae. Several studies have shown that expression of heterologous genes in some cases can be largely increased (15). The effect of the codon optimization could potentially be measured by comparing expression levels of the optimized and the original gene.

Fig 4: - Working hard on our transformants.

Cell wall structure and GfaA

The choice of building materials is always based on a set of parameters like strength, weight, thermal conductivity, durability, and price. Fungal materials are typically lightweight, good thermal insulators, have a good compressive strength (16), and in the context of Martian settlement, they could likely be made comparatively cheaper than building materials brought from Earth. Long-term durability is still somewhat an open question - especially in the context of Martian use. Biodegradability, which is a potential issue on Earth, is not a significant problem on Mars due to its extreme environment, thus suggesting that long-term stability might be achievable.

Fungal materials have shown to be sufficiently strong to make structures and various fibreboards. Given the lower gravity on Mars, fungal structures should be able to sustain themselves. We have models which suggest that fungal materials might also be able to sustain the internal pressure of habitats, yet it seems that making the materials stronger would be a desirable phenotypic characteristic to attain. Research suggested that the gene gfaA might be of interest having materials in mind. The structural strength of fungal cells walls comes from $\beta$-Glycans and chitin. gfaA encodes for a fructose-6-phosphate transferase that catalyzes the first and rate-limiting step in the UDP-N-acetylglucosamine biosynthetic pathway, the immediate precursor of chitin (17).
Studies have shown that after induced cell wall stress in A. niger, there are higher levels of gfaA mRNA present and that this is accompanied with higher levels of chitin deposition in the cell wall (17). We theorized that over-expression of this enzyme in Aspergillus oryzae might lead to a higher-than-normal chitin content of the fungal cell walls and hence, hopefully, stronger materials.

Colors and selectable markers - AmilCP

Finally, we have also been working on with the amilCP gene. amilCP encodes a blue chromoprotein and exhibits strong blue color when expressed in E. coli, and we wanted to use amilCP constructs as a reporter system to test transformation success of different fungi.

Mission Architecture

So how do we propose going about this entire endeavor? When a finalized version of our GMO fungus has been produced, the idea for astronauts is to bring a container with our fungus spores and a container with cyanobacterium to Mars. On their way, the astronauts will cultivate cyanobacteria in a growing vessel that is shaped like a long triangle. As you can see on the cross-section drawing, the vessel is a lightweight, collapsible container that can be fitted with a gas pump (pump not included in Fig(5)). The sides are zippable and on the inside, they are sown in spacers that make the inside volume of the vessel carve out our brick. The cyanobacteria will fill out the volume of the growing vessel while being fed CO2 through a pump, into the vessel. When the growing vessel is filled with the bacterium, the astronauts will put the GMO fungal spores on top of the bacteria. After a short while, the fungi will have consumed the cyanobacteria and filled out the entire volume of the vessel. The astronauts can then open and start building their habitat from fully formed fungal bricks.

Fig 5: The figure illustrates the growth vessels that the astronauts could utilise to grow shaped building material from fungus. They start by cultivating Cyanobacteria,. Hereafter, they plant fungal spores into the bacterium, letting it grow into fully formed fungi bricks.

(1)Williams M. 2017. What is the Weather like on Mars?.

(2)Verseux C, Baqué M, Lehto K, De Vera JPP, Rothschild LJ, Billi D. 2016. Sustainable life support on Mars - The potential roles of cyanobacteria. Int J Astrobiol 15:65–92.

(3)Egan M, Kreso J. 2011. Expediting Factors in Developing a Successful Space Colony. Worcester Polytechnic Institute, Massachusetts.

(4)Wolchover N. 2012. What are the Limits of Human Survival?. Live Science.

(5)Walter T. Oxygen and Human Requirements.

(6)Williams DR. 2018. Mars Fact Sheet.

(7)Andre M, Massimino D. 1992. Growth of Plants at Reduced Pressures: Experiments in Wheat - Technological Advantages and Constraints. Adv Space Res 12:97-106.

(8)Caceres PG. 2009. IMoM-6A. PDF.

(9)Zip Tie Domes. 2018. What is Geodesic Dome Frequency? An Explanation.

(10)Williams M. 2016. How bad is the radiation on Mars?. Universe Today.

(11)Catling DC, Cockell CS, McKay CP. Ultraviolet Radiation on the Surface of Mars. NASA Ames Research Center.

(12)Geng J, Yu SB, Wan X, Wang XJ, Shen P, Zhou P, Chen XD. 2008. Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system. J Biochem Biophys Methods 70:1151–1155.

(13)Tokyo Tech iGEM Team. 2009. Protection from UV ray.

(14)Kanehisa Laboratories. 2018. Tyrosine Metabolism - Aspergillus oryzae.

(15)Zhou Z, Dang Y, Zhou M, Li L, Yu C, Fu J, Chen S, Liu Y. 2016. Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proc Natl Acad Sci 113:E6117–E6125.

(16)Yang Z (Joey), Zhang F, Still B, White M, Amstislavski P. 2017. Physical and Mechanical Properties of Fungal Mycelium-Based Biofoam. J Mater Civ Eng 29:04017030.

(17)Ram AFJ, Arentshorst M, Damveld RA, vanKuyk PA, Klis FM, van den Hondel CAMJJ. 2004. The cell wall stress response in Aspergillus niger involves increased expression of the glutamine: Fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall. Microbiology 150:3315–3326.

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-<p>Engineering fungal mycelium to create viable building materials for on Mars is not a small challenge. This project, which was inspired by conversations with Lynn J. Rothschild from NASA, whom we owe thanks both for the inspiration and subsequent sparring on ideas, has relied on a several scientific fields to try to tackle the problems we have found along the way.
+<p style="text-align:justify">Engineering fungal mycelium to create viable building materials for Mars is not a small challenge. This project, which was inspired by conversations with Lynn J. Rothschild from NASA, and the Stanford-Brown-RISD iGEM team, whom we owe thanks to both for the inspiration and subsequent sparring on ideas, has relied on several scientific fields to try to tackle the problems we have found along the way. Being inspired by the idea of simple and cost-effective construction on Mars, our design focuses on the fungal aspect of how a cyanobacteria/fungal production system can be used to create building materials. As we explored this idea, we were presented with many problems to be solved and even more, questions to be answered. <br>The first and most pressing one, how does one cultivate a fungus on Mars? Moreover, which fungal species should we use? Can we determine and model the growth characteristics of our fungi? Is the strength of fungal materials determined by any distinct genes and is it possible to regulate these in a way that will make our materials even tougher? And not least, how do we make the system cost-effective? We set out to answer these questions to guide the creation of our final design.
-Our design inspired by the idea of simple and cost-effective construction on Mars, which its simplest terms says that instead of spending millions if not billions on transporting the materials needed for habitats to Mars, one could instead bring a set of vials: One vial with a cyanobacteria, which will harness the carbon in the atmosphere and the oxygen in the martian ice to create biomass that can be used as ‘substrate’ for fungal cultures to create range of biomaterials. Our project focussed on the fungal aspect of such a system and how it could be used to create building materials for habitat construction on-site. This exciting challenge raises many questions that have to be answered and even more problems have to be dealt with. First off, how does one cultivate fungi on Mars? Moreover, Which fungal species should we use? How do we make the system cost-effective? Are the strength of fungal materials determined by any distinct genes and it is possible to regulate these in a way that will make our materials even tougher?
-We set out to answer these questions, the answers of which guided the creation of our final design</p>
+<div id="growingfungusonMars">
 </div>
-<div class="frontspace">
-<h2>Building Buildings on Mars</h2>
 </div>
-<div class="col-xs-12">
-<div class="verticalLineright textbreather  verticalrightscience">
-<div class="interlabspace">
-<h3 style="text-align: right;margin-bottom: 35px; color:#0C233F;">How to build buildings on Mars</h3>
-<p>When the first Martian settlers arrive, They’ll want to erect livable habitats; buildings. When using conventional construction methods, they’d be restricted to either having to use heavy machinery in order to move local building materials, or having to bring the construction materials with them. Both options are an expensive cut in the weight and volume budget of any space mission, where, especially, mass is the key parameter of the mission cost.<br>
-This Projects’ novel approach is for the astronauts to pack a bag with fungal spores, in order for them to bring a super lightweight building material that is able to quickly grow into any shape by the aforementioned procedure in the first section.<br>
-An obvious question is then: What physical parameters will the fungal bricks have to withstand, for them to be used as building material on Mars? Initially, constructed as a building, they’d have to be able to contain a livable pressures. The average pressure on earth is 1.013 bar with 21% oxygen. The lowest “human life sustaining” pressure is 0.121 bar, assuming a 100% oxygen concentration. The average Martian pressure is 0.006 bar. This is a meere 0.6% of the average barometric pressure on earth.
-What building design/geometry is the best at containing pressures? Let’s investigate a dome design and a box design.
-  </p>
+<div class="verticalLineright textbreather interlabspace verticalrightyellow ">
+<h2 style="text-align: right;margin-bottom: 35px; color:#D8A764;">Growing Fungus on Mars</h2>
-</div>
+<p style="text-align:justify">
+Growing live fungus on Mars will be challenging as the environment on Mars is extremely hostile compared to Earth. In addition to the extreme cold, the atmosphere is very thin at less than 1% of the density of Earth’s atmosphere and consists of 96% carbon dioxide and virtually no oxygen (1). Moreover, our fungus needs organic compounds as a source of carbon and energy. Bringing substrates from Earth is out of the question due to the costs of transportation, thus <i>in situ</i> production of materials is vital. To solve the problem of available carbon sources, one can introduce cyanobacteria to the production system. Cyanobacteria can utilize the carbon dioxide in the Martian atmosphere, albeit, like the fungus, they, too, are unable to grow directly in the extreme conditions of Mars. To solve the problem of the Martian hostility it is necessary to have a production facility. Such a production facility for cyanobacteria has been theorized by researchers at NASA (2). Using covered thermally insulated ponds with compressed air and water from the Martian ice, it would be possible to grow cyanobacteria on Mars. Supporting this idea is the fact that all of the essential elements needed for cyanobacterial and even plant growth can be found on Mars (2). Based on this system theorized by NASA, we suggest appending it with the capabilities of using the cyanobacterial biomass to produce fungal materials.
+<br><br>
-<div style="display:block">
+<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/8/8f/T--DTU-Denmark--Design2.jpg" style="width: 60%;"> </p><figcaption><p style="text-align:center; font-size:14px;">Fig 1: - Creating bricks in unconventional molds. </p></figcaption>
-<div class="col-xs-5">
+</p>
-<img src="https://static.igem.org/mediawiki/2018/1/10/T--DTU-Denmark--deisgn1.png" style="width: 90%;"  >
-<figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1: </b> - Dome structure<br>Volume: 30m<sup>3</sup><br>Area: 48.6 m<sup>2</sup></p></figcaption>
+<p style="text-align:justify">
+However, non-destructive ways of harvesting nutrients could possibly lead to a more effective process. So instead of using lysed-dead cyanobacteria, there is also the possibility of using biomass produced by them as a secreted substrate. Cyanobacteria can provide heterotrophic organisms with both nitrogen and oxygen through their photosynthetic and nitrogen-fixing pathways. Specifically, they can convert carbon dioxide, the most abundant gas in Mars’ atmosphere, into oxygen and at the same time provide NH<sub>4</sub><sup>+</sup>  to the feeding organisms by utilizing N fixation (2). These problems are beyond the scope of our project, but provides an interesting starting point for future projects.
-</div>
+  </p>
-<div class="col-xs-5">
-<img src="https://static.igem.org/mediawiki/2018/1/16/T--DTU-Denmark--design2.png" style="width: 90%;">
-<figcaption style="text-align:center; font-size:14px;"><p style="text-align:center; font-size:14px;"><b>Fig. 2: </b> - Box structure<br>Volume: 30m<sup>3</sup><br>Area: 62 m<sup>2</sup></p></figcaption>
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 </div>
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+<div class="verticalLine interlabspace textbreather verticalleftred ">
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+<h3 style="text-align: left;margin-bottom: 35px; color:#AD2D00;">Choice of Organism</h3>
-<p>
-Based on this small review, using an arbitrary volume, it was concluded that the dome is the superior design for the building geometry. Reasons are as follows: It uses less building material per volume, the strength demand is lower in the dome because of the force build-up in the corners in the box; demanding a stronger brick material for the box design. The dome is as a whole intrinsically better at distributing the forces throughout the structure. The dome can be constructed from a singular unitary brick design; the triangle. <br><br>
-A dome can be constructed from a mix between hexagons and pentagons, but it turns out that if the dome is constructed from triangles, it is stronger and it is more convenient to only need to bring one mold for the bricks.
-After having sifted through the literature, it was decided that we are aiming to build and simulate a 3v dome. This means that the dome is constructed from two kinds if triangles with only slightly different side lengths. the combination of side lengths are as follows: AAB and BCC, where each letter denotes a distinct length. Our Final Brick design was based on these triangles with a meshing pattern, so that the triangles wouldn’t need additional materials to hold them together. They do it by utilising friction fit. When a dome has been erect, it would be sprayed with some curing agent, making the structure air-tight.
+<p style="text-align:justify">The choice of organism used in our project was based on an inductive strategy. We had two variables needed to be fulfilled in our attempt to achieve the goals we had set. The first was to find a fungus species that, given the right substrate, would have the ability to form firm brick structures. The second was to use a safe microorganism with a sequenced genome and established genetic methods, thus enabling us to do genetic modifications. The project started with three candidate-organisms: <i>Pleurotus ostreatus</i>, <i>Schizophyllum commune</i> and <i>Aspergillus oryzae</i>. These organisms have previously been grown on a variety of substrates and some have been used for creating bricks. We were also recommended to use a <i>Fusarium</i> by <a href="https://www.novozymes.com/en" target=”"_blank">researchers at Novozymes</a>, but opted to focus on the ones we had multiple recommendations on.  For various reasons, our choice ended up being <i>A. oryzae</i>; <i>A. oryzae</i> is comparatively easy to grow on various substrates, it grows fast, gives consistently good results and is a well-established model organism. Though the other fungal species have previously shown to have desirable characteristics, when it comes to creating fungal materials, the <a href="https://2018.igem.org/Team:DTU-Denmark/Results-choosing-organism" target="_blank">results</a> of our initial experiments made us decide on doing our research in <i>A. oryzae</i> with the possibility of transferring the discoveries to other fungal species at a later point in the project.
 </p>
-</div>
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-<div class="col-xs-6">
-<img src="https://static.igem.org/mediawiki/2018/f/f7/T--DTU-Denmark--design-dome-1.jpg" style="width: 90%;"  >
-<figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 3: </b> - Triangle sizes</p></figcaption>
+<div id="buildstructures">
 </div>
-<div class="col-xs-6">
-<img src="https://static.igem.org/mediawiki/2018/0/06/T--DTU-Denmark--design-dome-2.jpg" style="width: 90%;">
-<figcaption style="text-align:center; font-size:14px;"><p style="text-align:center; font-size:14px;"><b>Fig. 4: </b> - Triangle dome structure</p></figcaption>
-</div>
 </div>
-</div>
-</div>
-</div>
+<div class="verticalLineright textbreather interlabspace verticalrightdarkblue">
-<div class="col-xs-12">
-<div class="interlabspace">
-<h3>Determining fungal material properties</h3>
+<h3 style="text-align: right;margin-bottom: 35px; color:#001D43;">How to Build Structures on Mars</h3>
-<p>When determining material properties, experiments are critical. Depending on how many parameters you want to examine, the number of experiments tend to exponentially increase. Using Fedorov Exchange Algorithms we were able to reduce our experiment-space (number of experiments), in the first run, from 216 to 64 and in the second run from 648 to 68, which, as you can imagine, is a huge time saver.<br><br>
+<p style="text-align:justify">
-It became clear to us that we needed to produce a lot of fungi sample/”cubes”. <br>
+When wanting to erect livable habitats on Mars made of fungal materials, an obvious question is: What physical parameters will the fungal bricks have to withstand for them to be used as building material on Mars? Martian gravity is about 38% of Earth’s (3) makes it easier to erect self-sustainable structures on Mars than on Earth. However, the greater challenge is enabling structures to sustain the internal pressure generated by the pressure difference between the liveable habitat and that of the Martian atmosphere. The average pressure on Earth is 1.013 bar with 21% oxygen. The lowest human life-sustaining pressure is 0.121 bar, assuming a 100% oxygen concentration (4). Though, this is not desirable due to extreme fire hazards. At these oxygen concentrations, everything becomes combustible. Near the lower border of what is safe/healthy and sustainable for human life is an atmosphere containing 19.5% oxygen at approximately 0.65 bar (5). As stated earlier, the average Martian pressure is 0.006 bar. This is only 0.6% of the average barometric pressure on earth (6). <br><br>
-Initially, we fabricated some acrylic boxes. These were fast and easy to make. It turns out that acryl does not withstand the pressures and temperature when the fungi gets autoclaved. We turned to metal. These metal boxes took a long time to make, and after having fabricated 11, it was decided that the effort would not be worth it. After having done some research, silicone ice cube trays turned out to be the perfect alternative to our metal boxes. Silicone can easily withstand autoclaving and allowed for very uniformly shaped samples. <br><br>
-The test samples/ fungi cubes were all prepared in slightly different ways, according to our experiment optimization: Baking times, compound composition, substrates etc. All the samples were subjected to a destructive compressional test, where the stress/strain-curve was determined for compressional strength. In order to obtain the Young’s Modulus of the fungi, the tensile strength would also have to be measured. When you know Young’s Modulus, you have the insights of the entire materials’ properties and characteristics.
-</p>
+What building design/geometry is the best at containing pressures? For simulation purposes, the internal pressure is 1 atmosphere. Humans can live at lower pressures, but lab-equipment and plant growth are sensitive to lower pressures (7). This is why the International space station maintains an internal pressure of 1 ATM. Looking at a dome and a box design, the COMSOL simulations on Fig(2) and Fig(3), strongly indicate, that the dome design is the better choice. We assume that: Equal internal volume, an internal pressure of 1 bar and an external pressure of 0.006 bar. In order for the box, to be able to contain this pressure, the wall thickness would have to be 1.3 m thick, compared to the domes 0.3 m wall thickness. The pressure distribution is also clearly more uniformly distributed in the dome, compared to the box. The relations between “volume to area” of the dome and box are as follows:<br>
+\begin{equation}
+Area_{circle} = r^2 \cdot \pi
+\end{equation}
+\begin{equation}
+Area_{dome} = Area_{circle} + \frac{r^2 4 \pi}{2} = 3 r^2
+pi
+\end{equation}
-</div>
+\begin{equation}
+Area_{box} = 2a^2 + 2a^2 + 2a^2 =6 a^2
+\end{equation}
-</div>
+\begin{equation}
+\frac{Area}{Volume} -relations :
+\end{equation}
+\begin{equation}
+\frac{a^3}{Area_{box}}  = a \cdot \frac{1}{6}
+\end{equation}
+\begin{equation}
+\frac{\frac{4}{3} \pi r^ 3}{Area_{dome}} = \frac{2}{9} \cdot r
+\end{equation}
+What becomes apparent from this, is that the “volume to area”- relation is lower for a box. This means that you would need more building material to construct a box, compared to a dome of equal volume.
-<div class="col-xs-12">
+<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/0/0b/T--DTU-Denmark--minimum_thickness.png" style="width: 100%;"> </p><figcaption><p style="text-align:center; font-size:14px;">Fig 2: - The picture shows how the internal stresses accumulate in a dome, simulated in COMSOL. It also shows the minimal wall thickness (0.3 m) when using Aspergillus as building material in Mars, if the dome has to be able to contain 1 bar. </p></figcaption>
-<div class="interlabspace">
+</p>
-<h3>How to grow fungi on Mars</h3>
-<p>
-Growing living organisms and specifically fungi extraterrestrially can be challenging, as the surface of Mars is really hostile. In addition to the extreme cold, there is little atmosphere to speak of and virtually no oxygen. In our attempt to tackle these challenges we aim to introduce cyanobacteria in these extreme conditions.<br><br>
-To start with, cyanobacteria are thought to have played a pivotal role in the evolution of Earth’s atmosphere. Their biological functions are facilitated by producing oxygen, nutrients and vitamins through photosynthesis which in turn can led in to the formation of a human-friendly environment. Therefore, our general plan is to use cyanobacteria which would convert the ample supplies of Mars’ CO² into oxygen, reducing carbon dioxide and at the same time making human occupancy a valid possibility. Seeding Mars’ atmosphere with cyanobacteria, can lead to the conversion of its most abundant gas into something breathable for humans which will at the same time serve as a growing substrate for living organisms.<br><br>
-We hope that one day, manned missions will be able to take advantage of the oxygen created by these bacteria by either combining it with nitrogen to create breathable air or by using it for consumption over and over again.
+<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/f/f3/T--DTU-Denmark--box_1atm_1.347m.png" style="width: 100%;"> </p><figcaption><p style="text-align:center; font-size:14px;">Fig 3: - The internal stress accumulation of a box is a lot higher than a dome. In order for an box, of same volume as Fig(2), the wall thickness would have to be 1.3 m in order to contain 1 bar against the Martian atmosphere.</p></figcaption>
 </p>
+<div id="improvingfungi">
 </div>
 </div>
@@ Line 157: / Line 137: @@
 <div class="col-xs-12">
-<div class="interlabspace">
+<div class="verticalLine interlabspace textbreather verticalleftyellow">
-<h3>Choice of organism</h3>
+<h3 style="text-align: left;margin-bottom: 35px; color:#D8A764;">Improving the fungal materials through synthetic biology</h3>
-<p>
+<p style="text-align:justify">
-The choice of organism used in our project was based on an inductive strategy. We had two variables needed to be fulfilled in our attempt to achieve the goals that we had set. The first one was to find a fungi species that when given the right substrate would have the ability to form firm brick structures. The second one was to use a microorganism that would have its genome sequenced and thus enabling it to withstand genetic modifications. The project started with three candidates-organisms: Pleurotus Ostreatus, Aspergillus Oryzae and Schizophyllum Commune. Our choice ended up being relatively simple as the results taken from every candidate except from A.oryzae, were off the mark compared to our initial expectations. Briefly, Aspergillus oryzae when given rice as a substrate could form strong and firm bricks accompanied with swift spore formation. A possible explanation of this feature could be that Aspergilli can release a variety of enzymes that are capable of breaking down their substrate into simpler compounds, absorbing them through their vegetative hyphae. The hyphae when being in a favorable environment (moisture, warmth and nutrients) allows the fungi to grow, spread and continue reproducing across the surface of the substrate and eventually taking the shape of the engulfing container.<br><br>
+When using fungal mycelium and organic materials as the basis for building materials, a new venue of possibilities suddenly appear. Fungi are genetically modifiable. This allows for tuning of different external parameters or even adding properties such as biosensors etc. Thus, the interesting question to answer in this aspect is; what genetic modifications are beneficial when it comes to construction on Mars?<br><br>
-Furthermore, Aspergillus oryzae belongs to a family of fungi that has been used vastly in a lot of aspects of the industry. Species belonging in the Aspergillus family, prosper in a variety of different climates around the world and have a variety of different applications ranging from microbial fermentations to medicinal applications. What is more, Aspergilli have low pathogenic potential as they do not produce aflatoxins or any other cancerogenic metabolites, making the conduction of safe laboratory experiments possible.
+Like any other organism, any fungus is optimized for the survival of its species in its niche. No fungal strain is solely optimized for creating strong materials, as that would not be an advantageous evolutionary strategy. By making the fungus a little worse at surviving, we aim to make it better for a building. In our project, we ended up focusing primarily on two genes; <i>melA</i> and <i>gfaA</i>.
 </p>
 </div>
+<div id="protectionfromuv">
+</div>
 </div>
@@ Line 177: / Line 163: @@
 <div class="col-xs-12">
-<div class="interlabspace">
+<div class="verticalLineright textbreather interlabspace verticalrightred">
-<h3>Improving the fungal materials through synthetic biology</h3>
-<p>
-When using fungal mycelium and organic materials as the basis for your building materials, a new venue of possibilities suddenly appear. The fungi is genetically modifiable, which allows for tuning of different external parameters or even adding properties such as biosensors etc.
-The interesting question to answer is this respect is: What genetic modifications are beneficial when it comes to construction on Mars?<br><br>
-Like any other organism, any fungi is optimized for the survival of its species in its niche. No fungal strain is solely optimized for creating strong materials, as that wouldn’t be a advantageous evolutionary strategy. By making the fungi a little worse at surviving we aim to make it better at building<br><br>
-Understanding morphology and the environment under which it lives is pivotal for identifying potential gene candidates<br>
+<h3 style="text-align: right;margin-bottom: 35px; color:#AD2D00;">Protection from UV radiation and Melanin</h3>
-(Briefly on fungal morphology, The strength of fungal cell walls is provided by its a)<br>
+<p style="text-align:justify">
+One part of what makes the surface of Mars a hostile environment is its radiation. Unlike the terrestrial atmosphere, the Martian atmosphere does not shield well against cosmic radiation, making radiation a serious concern on the red planet (10). Part of the radiation issue is short wavelength UV radiation such as UVB and UVC, which is not just harmful to living tissue but also causes radiation damage to solid materials (11).
+Nature has evolved systems that give the protection against UV radiation, most well-known is the pigment melanin. Overexpressing melanin might make fungal materials more resilient against radiation damage. Studies have shown that melanotic bacteria experience increased protection from the full spectrum of UV compared to counterparts without melanin (12).
-In our project we ended up focussing especially two genes, melA and gfaA.
+To slow down the potential erosion from UV radiation, we wanted to express the gene <i>melA</i> from <i>Rhizobium etli</i> CFN42 (13) in our fungal host. A previous iGEM team from the Technical Institute of Tokyo have shown that overexpression of melanin in <i>E. coli</i> can been achieved using the <a href="https://2018.igem.org/Team:DTU-Denmark/Results-melA"><i>melA</i> gene</a>. <i>melA</i> encodes this for a tyrosinase that catalyses the enzymatic steps converting tyrosine into dopaquinone. We investigated the biosynthetic pathway of melanin in <i>A. oryzae</i> and discovered the same metabolic step as encoded for by <i>melA</i>  (14). Heterologous expression of MelA in A. oryzae may thus also increase the production of melanin.
@@ Line 196: / Line 179: @@
 </div>
-</div>
+<div id="melanin">
+</div>
+</div>
-<div class="col-xs-12">
-<div class="interlabspace">
-<h3>Protection from UV radiation and Melanin</h3>
-<p>
-One part of what makes Mars as harsh environment is radiation. Unlike the terrestrial atmosphere, the martian atmosphere does not shield well against cosmic radiation. Among such kind of radiation in UV light (higher amount of damaging UVC and UVB radiation on surface. (https://mars.jpl.nasa.gov/mgs/sci/fifthconf99/6128.pdf))
-Melanotic bacteria increased protection for the full spectrum of UV (We want to transfer this. (https://www.sciencedirect.com/science/article/pii/S0165022X0700190X?via%3Dihub)<br><br>
-“melA encodes for the first step in the melanin biosynthetic pathway. it encodes a tyrosinase that catalyzes the reaction dopaquinine”.
-Using the tokyo tech melA gene (https://2009.igem.org/Team:Tokyo_Tech/BlackenedEcoli#Protection_from_UV_ray) (refitted to fungi).<br><br>
-Melanin is a pigment and a well-known UV-protectant. It is a senseful choice both in the sense it fits our theme and furthermore it gives us a selectable genotype. By UV-irradiating the transformants vs. non-transformants it will be possible to show the effect of melanin on the growth rate/or ability to grow.<br><br>
-Melanin thermally stable (https://pdfs.semanticscholar.org/42b5/d1c95260addbfdf54a8632a771f16f43c40f.pdf)
-</p>
-</div>
-</div>
@@ Line 234: / Line 197: @@
 <div class="col-xs-12">
-<div class="interlabspace">
+<div class="verticalLineright textbreather interlabspace verticalrightyellow">
-<h3>Melanin (Codon optimized for <i>A. oryzae</i>)</h3>
-<p>
+<h3 style="text-align: right;margin-bottom: 35px; color:#D8A764;">Melanin (Codon optimized for <i>A. oryzae</i>)</h3>
-(Codons is a key concept when it comes to working with fungi, and a wisely codon optimized gene for a specific species of fungi and see yield increases on heterologous genes by factors - e.g: http://www.pnas.org/content/113/41/E6117 ). Expressing the codon optimized gene alongside the original gene will show the relative expression level and thus the effect of codon optimization.
+<p style="text-align:justify">
+As a part of the project we set out to codon optimize, the Tokyo Tech <i>melA</i> gene from <i>Rhizobium etli</i> CFN42 to increase the expression in <i>A. oryzae</i>. Several studies have shown that expression of heterologous genes in some cases can be largely increased (15). The effect of the codon optimization could potentially be measured by comparing expression levels of the optimized and the original gene.
+<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/f/f4/T--DTU-Denmark--Design1.jpg" style="width: 60%;"> </p><figcaption><p style="text-align:center; font-size:14px;">Fig 4: - Working hard on our transformants. </p></figcaption>
 </p>
-</div>
+</p>
+</div>
+<div id="cellstructure">
+</div>
 </div>
@@ Line 250: / Line 217: @@
+<div class="col-xs-12">
+<div class="verticalLine interlabspace textbreather verticalleftred">
-<div class="col-xs-12">
+<h3 style="text-align: left;margin-bottom: 35px; color:#AD2D00;">Cell wall structure and GfaA</h3>
-<div class="interlabspace">
-<h3>Cell wall structure and GfaA</h3>
+<p style="text-align:justify">
+The choice of building materials is always based on a set of parameters like strength, weight, thermal conductivity, durability, and price. Fungal materials are typically lightweight, good thermal insulators, have a good compressive strength (16), and in the context of Martian settlement, they could likely be made comparatively cheaper than building materials brought from Earth. Long-term durability is still somewhat an open question - especially in the context of Martian use. Biodegradability, which is a potential issue on Earth, is not a significant problem on Mars due to its extreme environment, thus suggesting that long-term stability might be achievable.<br><br>
-<p>
+Fungal materials have shown to be sufficiently strong to make structures and various fibreboards. Given the lower gravity on Mars, fungal structures should be able to sustain themselves. We have models which suggest that fungal materials might also be able to sustain the internal pressure of habitats, yet it seems that making the materials stronger would be a desirable phenotypic characteristic to attain. Research suggested that the gene <i>gfaA</i> might be of interest having materials in mind. The structural strength of fungal cells walls comes from $\beta$-Glycans and chitin. <i>gfaA</i> encodes for a fructose-6-phosphate transferase that catalyzes the first and rate-limiting step in the UDP-N-acetylglucosamine biosynthetic pathway, the immediate precursor of chitin (17).<br>
-GfaA was chosen having structural strength of the materials in mind. The structural strength fungal cells walls come from β-Glycans and Chitin, (Chitin being the most important of the two).  Gfa encodes for an enzyme ??? that catalyzes the rate limiting step in the production of chitin, and overexpression. (mutants overexpressing Gfa in yeast have been shown to have I higher wall chitin content) and we theorized that overexpression of this enzyme in aspergillus oryzae might lead to a higher-than-normal Chitin content of the fungal cell walls and henceforth hopefully stronger materials
+Studies have shown that after induced cell wall stress in <i>A. niger</i>, there are higher levels of <i>gfaA</i> mRNA present and that this is accompanied with higher levels of chitin deposition in the cell wall (17).  We theorized that over-expression of this enzyme in <i>Aspergillus oryzae</i> might lead to a higher-than-normal chitin content of the fungal cell walls and hence, hopefully, stronger materials.
-rate limiting - transferase , etc...<br>(http://mic.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.27249-0#tab2).
-Using Hph or NAT1 as a selection marker.
@@ Line 265: / Line 232: @@
 </p>
 </div>
+<div id="selectablemarkers">
+</div>
 </div>
@@ Line 272: / Line 240: @@
 <div class="col-xs-12">
-<div class="interlabspace">
+<div class="verticalLineright textbreather interlabspace verticalrightdarkblue">
-<h3>Colors and selectable markers - AmilCP</h3>
-<p>
+<h3 style="text-align: right;margin-bottom: 35px; color:#001D43;">Colors and selectable markers - AmilCP</h3>
-AmilCP is a blue Chromoprotein.
-We wanted to use the expression of a color protein as a “Hello World” gene. AmilCP is a blue color protein? (pigment?), and by such can be used as an easily distinguishable marker.
+<p style="text-align:justify">
+Finally, we have also been working on with the <a href="https://2018.igem.org/Team:DTU-Denmark/Results-amilCP"><i>amilCP</i> gene</a>. <i>amilCP</i> encodes a blue chromoprotein and exhibits strong blue color when expressed in <i>E. coli</i>, and we wanted to use  <i>amilCP</i> constructs as a reporter system to test transformation success of different fungi.
@@ Line 284: / Line 252: @@
 </p>
+</div>
+<div id="missionarchitecture">
 </div>
@@ Line 292: / Line 263: @@
 <div class="col-xs-12">
-<div class="interlabspace">
+<div class="verticalLine interlabspace textbreather verticalleftyellow">
-<h3>Mission Architecture</h3>
+<h3 style="text-align: left;margin-bottom: 35px; color:#D8A764;">Mission Architecture</h3>
-<p>
+<p style="text-align:justify">
-So how do we propose going about this entire endeavor?  When a finalized version of our GMO fungi has been produced, the idea for astronauts to bring a container with our fungi spores and a container with cyanobacterium. These are brought to Mars. On their way, the astronauts will cultivate the cyanobacteria in a growing vessel, which is shaped like a long triangle. As you can see on the cross-section drawing, the vessel is a lightweight, collapsible container that can fitted with a gas pump. All the sides are zippable and on the inside, there are sown in spacers that makes the inside volume of the vessel, carve out our brick. The cyanobacteria will fill out the volume of the growing vessel while being fed CO2 through a pump, into the vessel. When the growing vessel is full with the bacterium, the astronauts will plant the GMO fungi spores onto the bacteria. After a short while the fungi will have “eaten” the cyanobacteria and taken its place, filling out the entire volume of the vessel. The astronauts can then open and start building their habitat from fully formed fungi bricks.
+So how do we propose going about this entire endeavor?  When a finalized version of our GMO fungus has been produced, the idea for astronauts is to bring a container with our fungus spores and a container with cyanobacterium to Mars. On their way, the astronauts will cultivate cyanobacteria in a growing vessel that is shaped like a long triangle. As you can see on the cross-section drawing, the vessel is a lightweight, collapsible container that can be fitted with a gas pump (pump not included in Fig(5)). The sides are zippable and on the inside, they are sown in spacers that make the inside volume of the vessel carve out our brick. The cyanobacteria will fill out the volume of the growing vessel while being fed CO2 through a pump, into the vessel. When the growing vessel is filled with the bacterium, the astronauts will put the GMO fungal spores on top of the bacteria. After a short while, the fungi will have consumed the cyanobacteria and filled out the entire volume of the vessel. The astronauts can then open and start building their habitat from fully formed fungal bricks.
-<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/8/8c/T--DTU-Denmark--design-ma.png" style="width: 70%;"> </p>
+<p style="text-align:center"><img src="https://static.igem.org/mediawiki/2018/c/c4/T--DTU-Denmark--GrowthMissionArc.png" style="width: 100%;"> </p><figcaption><p style="text-align:center; font-size:14px;">Fig 5: The figure illustrates the growth vessels that the astronauts could utilise to grow shaped building material from fungus. They start by cultivating Cyanobacteria,. Hereafter, they plant fungal spores into the bacterium, letting it grow into fully formed fungi bricks.  </p></figcaption>
+</p> </p>
@@ Line 314: / Line 286: @@
+</div>
+<p style="color:#000; font-size:14px;">
+(1)Williams M. 2017. What is the Weather like on Mars?.<br><br>
+(2)Verseux C, Baqué M, Lehto K, De Vera JPP, Rothschild LJ, Billi D. 2016. Sustainable life support on Mars - The potential roles of cyanobacteria. Int J Astrobiol 15:65–92.<br><br>
+(3)Egan M, Kreso J. 2011. Expediting Factors in Developing a Successful Space Colony. Worcester Polytechnic Institute, Massachusetts.<br><br>
+(4)Wolchover N. 2012. What are the Limits of Human Survival?. Live Science. <br><br>
+(5)Walter T. Oxygen and Human Requirements. <br><br>
+(6)Williams DR. 2018. Mars Fact Sheet. <br><br>
+(7)Andre M, Massimino D. 1992. Growth of Plants at Reduced Pressures: Experiments in Wheat - Technological Advantages and Constraints. Adv Space Res 12:97-106. <br><br>
+(8)Caceres PG. 2009. IMoM-6A. PDF. <br><br>
+(9)Zip Tie Domes. 2018. What is Geodesic Dome Frequency? An Explanation. <br><br>
+(10)Williams M. 2016. How bad is the radiation on Mars?. Universe Today. <br><br>
+(11)Catling DC, Cockell CS, McKay CP. Ultraviolet Radiation on the Surface of Mars. NASA Ames Research Center. <br><br>
+(12)Geng J, Yu SB, Wan X, Wang XJ, Shen P, Zhou P, Chen XD. 2008. Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system. J Biochem Biophys Methods 70:1151–1155.<br><br>
+(13)Tokyo Tech iGEM Team. 2009. Protection from UV ray. <br><br>
+(14)Kanehisa Laboratories. 2018. Tyrosine Metabolism - Aspergillus oryzae. <br><br>
+(15)Zhou Z, Dang Y, Zhou M, Li L, Yu C, Fu J, Chen S, Liu Y. 2016. Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proc Natl Acad Sci 113:E6117–E6125.<br><br>
+(16)Yang Z (Joey), Zhang F, Still B, White M, Amstislavski P. 2017. Physical and Mechanical Properties of Fungal Mycelium-Based Biofoam. J Mater Civ Eng 29:04017030.<br><br>
+(17)Ram AFJ, Arentshorst M, Damveld RA, vanKuyk PA, Klis FM, van den Hondel CAMJJ. 2004. The cell wall stress response in Aspergillus niger involves increased expression of the glutamine: Fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall. Microbiology 150:3315–3326.
-</div>
+</p>
 </div>
 </div>
@@ Line 350: / Line 342: @@
 					<a href="https://2018.igem.org/Team:DTU-Denmark/Description">Project description</a>
 					&bull;
-                        <a href="https://2018.igem.org/Team:DTU-Denmark/Model">Modelling</a>
+                        <a href="https://2018.igem.org/Team:DTU-Denmark/Model">Modeling</a>
 					&bull;
 					<a href="https://2018.igem.org/Team:DTU-Denmark/Parts">Parts overview</a>

Difference between revisions of "Team:DTU-Denmark/Design"